To evaluate the T-SPOT.TB interferon-γ releasing assay and the tuberculin skin test (TST), for the diagnosis of latent tuberculosis infection(LTBI) and the development of subsequent active tuberculosis, in BCG-vaccinated HIV-infected individuals.HIV-infected individuals without clinical suspicion of active TB or a past history of TB were enrolled from 1 January 2008 to 30 November 2010. Both T-SPOT.TB test and TST were offered to the participants whom were followed up prospectively until April 30, 2012 for development of TB.Among the 909 participants, 25% had positive TST reactions with cut-off point of 5 mm and 15% had positive T-SPOT.TB results. After a median follow-up of 2.97 years, there were 5 cases developed culture-confirmed active TB , and the incidence was 0.17 per 100 person-years. The relative risks (RRs) for subsequent active TB in HIV-infected individuals with positive TST results, positive T-SPOT.TB results and dual positive results compared with the risk for individuals with negative results were 40.6 (95% CI 2.1–767.9), 73.9 (95% CI 3.9–1397.7) and 226.5 (95% CI 12.0–4284), respectively. The number needed to treat to prevent one subsequent TB case among patients with a positive TST, a positive T-SPOT.TB and dual positive results was 35, 22 and 8 respectively.Adopting positive results of the TST and T-SPOT.TB to screen LTBI among BCG-vaccinated HIV-infected individuals might be feasible. Number needed to treat for isoniazid preventive therapy could be reduced significantly by using dual positive strategy. Pneumocystis carinii pneumonia remained the most common opportunistic infections in PLHIV in Taiwan 5 in 2005 to 57/105 in 2010 HIV-infected individuals have a higher rate of progression from latent tuberculosis infection (LTBI) to active TB than do non-HIV-infected persons, even with the use of effective antiretroviral therapy and the relative risk reached ten-fold The tuberculosis skin test (TST) is the traditional method for the diagnosis of LTBI, but the test has low specificity due to cross reactions with the bacillus Calmette-Guérin (BCG) vaccine and non-tuberculous mycobacteria. Taiwan launched the neonatal BCG vaccination since 1965 and the coverage rate scaled up rapidly to over 80% since 1970 and continued above 97% after 2001 Mycobacterium tuberculosis antigens was developed recently and addresses many of the TST's limitations. One meta-analysis suggested that interferon-γ release assays (IGRAs) had higher specificity for TB infection than the TST does, especially in BCG-vaccinated, non-HIV infected populations A method based on the measurement of interferon-γ release by T cells incubated ex vivo with The objectives of this prospective, longitudinal study were to evaluate the T-SPOT.TB (an IGRA) and the TST, for the diagnosis of LTBI and the development of subsequent active tuberculosis, in BCG-vaccinated HIV-infected individuals. We tried to determine the clinical and epidemiological risk factors associated with positive results for the TST and the T-SPOT.TB test in HIV-infected individuals and recommend an appropriate protocol to better select HIV-infected individuals for IPT.3 are recommed to initiate antiretroviral therapy Participants were recruited on a voluntary basis from three HIV outpatient clinics  and two prisons (Taipei Prison and Taoyuan Prison) between January 1, 2008 and November 30, 2010. There was a total of 1000∼1200 HIV-infected patients followed up in the three HIV clinics regularly. Taipei prison accommodated 3,000 adult male inmates and around 200 among them were HIV-infected. Taoyuan prison accommodated 1,000 adult female inmates and around 150 among them were HIV-infected. According to Taiwan's regulation, inmates have been mandatorily tested for HIV upon entry to correctional facilities since 1990 The study was approved by the Centers for Disease Control, Department of Health, Executive Yuan, Taiwan; the Institutional Review Board (IRB) of Taipei Medical University Hospital and the IRBs at Taipei City Hospital and Taoyuan General Hospital. All participants gave written informed consent.Individuals aged 18 years and above and had HIV infection (confirmed by Western blot tests) were enrolled. Individuals with active TB or past history of TB were excluded. Written informed consents were provided to all participants before receiving screening tests. Individuals who understood the screening programme and who gave informed consent were enrolled. All potential participants who declined to participate or otherwise did not participate were eligible for treatment (if applicable) and were not disadvantaged in any other way by not participating in the study.Each patient's HIV-related information was collected by a review of medical records and from the Taiwan CDC HIV registry data. After consent was obtained, the standardized questionnaire was completed by all participants which including background sociodemographic and clinical information. The study nurse checked the presence of BCG scars prior to the examinations provided. The CD4+ lymphocyte count and HIV RNA load data were collected.M. tuberculosis collected for symptomatic patients were used to exclude active TB. The TST with purified protein derivative  and the T-SPOT.TB test  were done with the flow of drawing blood first followed by TST. The blood was processed by laboratory staff blind to the clinical status of the sample and interpreted according to the manufacturer's protocol. The transverse induration size of the TST was read by the nurses who administered the test between 48 and 72 hours after administration, induration size exceeding 5 mm was considered positive Baseline chest radiographs for each participant and sputum cultures for M. tuberculosis were routinely performed for symptomatic patients M. tuberculosis from sputum or other tissue.All patients, including those who declined treatment, were instructed on symptom-specific self-monitoring and reporting. Chest radiographs and sputum cultures for We evaluated the performance of TST and T-SPOT.TB with regard to the development of active TB in participants who did not receive IPT. Assuming a full protection of IPT, we will further calculate the number needed to treat to prevent subsequent TB disease for each test.Multivariate logistic regression was used for cross-sectional analysis with adjustment for all relevant potential confounders associated with positive TST, T-SPOT.TB and dual positive results. The concordance between the TST and T-SPOT.TB results was assessed using Cohen's kappa (κ) coefficient. The strength of the agreement was considered ‘poor’ for ≤0.20, ‘fair’ for 0.20<κ≤0.40, ‘moderate’ for 0.40<κ≤0.60, ‘substantial’ for 0.60<κ≤0.80 and ‘optimal’ for 0.80<κ≤1.00 P values were less than 0.05. Statistical analysis was performed with using SAS, version 9.2 software  and SPSS v 14.0 .One main outcome of interest was the incidence rate of active TB, stratified according to screening test and IPT use. To calculate the relative risks (RRs) of rare events, we used the Poisson distribution in survival analysis. The Kaplan-Meier survival analysis with a log-rank test was conducted for analysis of the effect of IPT. All statistical analyses were two-sided and were considered significant when the A total of 1,049 HIV-infected individuals were invited to complete the questionnaires for enrolment . The TSTFor the T-SPOT.TB test, 96.2% of the enrollees had a valid result, and 15% of them had positive results; the 38 patients (3.8%) with indeterminate results were similar to those with valid results in terms of TB risk factors and demographics.A total of 909 individuals had interpretable results for both the T-SPOT.TB test and the TST and were used for further analysis. Background characteristics of enrollees are summarized in p = 0.08. Repeat the analysis with or without prison stay revealed similar results for other covariates in the model.3 or without BCG vaccination, TST had significantly more positive results compared with T-SPOT.TB. The overall agreement between the two tests was only fair . The agreement and kappa values between the two tests was highest in individuals with CD4 cell counts <200 cells/mm3 , followed by individuals who had not received the BCG vaccine  and individuals aged 40 years or above .By using 10 mm or 15 mm as a cutoff of TST reaction, the agreement was only increased to 82% or 86% with the same kappa value. After a median follow-up duration of 2.97 years, 5 patients (0.55%) developed active TB were noted and the clinical information was presented in After exclude individuals with IPT, the TB incidences among HIV-infected individuals were stratified with a positive TST, a positive-SPOT.TB and dual positive results . Based oThere were only 87 (38%) of the 227 TST-positive subjects accepted IPT, with a treatment completion rate of 91.9%. Among patients with dual positive results, there were 4 active TB cases among 32 untreated patients, compared with 1 TB case among 49 patients taking IPT , the T-SPOT.TB test had a higher positive rate than the TST did, different from individuals with higher CD4+ lymphocyte counts. This finding suggested that the T-SPOT.TB test was less affected by CD4+ lymphocyte counts than the TST was in HIV-infected patients. But our sample size was small and more evidence was needed. Furthermore, though some guidelines recommend treat HIV-infected individuals with TST-negative/IGRA-positive results, there were no evidence shown that IPT could improve outcomes on this population The TB incidence among HIV-infected individuals was 0.17 per 100PY, around 2.7 times than general population in Taiwan, which was 62 per 100,000 population in 2008 Due to the low specificity, TST may not be a good tool to screen LTBI among BCG-vaccinated HIV-infected individuals, as BCG-vaccinated non-HIV individuals, which is the arduous situation we all faced in Asia One main goal of our study was to develop an appropriate protocol, instead of the current Taiwan's recommendation, to better select HIV-infected individuals for IPT thus to prevent subsequent TB. Unfortunately, there was insufficient evidence to conclude that T-SPOT.TB test was superior to the TST. And the majority of the individuals with positive TST or T-SPOT.TB results did not develop TB. There was only limited number of longitudinal studies focused on the application of IGRA and TST in the LTBI diagnosis among HIV-infected individuals and still no consensus 3), and 40% were on HAART; thus, our results may not be applicable to HIV-infected individuals with lower CD4+ counts or to those who experienced difficulty in obtaining HAART.Our study has important strengths and limitations. The study involved a prospective large cohort followed up, enabling us to assess the performance of available tests for LTBI diagnosis such as TST and IGRAs by using development of subsequent active TB as the endpoint. In low TB burden areas, such as developed countries, with TB incidences less than 20 per 100,000 person-years, it requires a huge sample size and a prolonged follow-up duration to achieve significant results. Thus there were only a limited number of studies available, especially among HIV-infected patients In conclusion, adopting positive results of the TST and T-SPOT.TB to screen LTBI among BCG-vaccinated HIV-infected individuals might be feasible. Number needed to treat for isoniazid therapy could be reduced significantly by using the dual positive strategy.


Dioscorea japonica Thunb. and Dioscorea nipponica Makino, was reported to have neurotrophic activity. Therefore, we investigated the therapeutic potential of DA-9801, in comparison with alpha lipoic acid (ALA), for peripheral nerves preservation in experimental diabetes. Experimental animals were divided into 4 groups, and each group was designated according to the type of treatment administered as follows: normal, DM, DM+DA-9801, and DM+ALA. After 16 weeks, response thresholds to tactile and thermal stimuli were higher in DM+DA-9801 group than in nontreated DM group. This degree of increase in DM+DA-9801 group indicates more therapeutic potency of DA-9801 than ALA. Western blot analysis showed more significant increase in NGF and decrease in TNF-α and IL-6 in DM+DA-9801 group than in DM or DM+ALA groups (P < 0.05). IENF density was reduced less significantly in the DM+DA-9801 group than in other DM groups . Mean myelinated axonal area in the sciatic nerves was significantly greater in DM+DA-9801 group than in other DM groups . Results of this study demonstrated potential therapeutic applications of DA-9801 for the treatment of diabetic peripheral neuropathy.DA-9801, a mixture of extracts from  Half of diabetic patients have chronic complication involving the peripheral nervous system, which results in diabetic peripheral neuropathy (DPN). Besides morbidity and mortality, DPN has diverse symptoms, which leads to the deterioration of the quality of life of diabetic patients , 2. DPN Dioscorea japonica known as SanYak and Dioscorea nipponica known as Buchema [DA-9801 is a mixture of extracts from  Buchema . Resultsn = 10 per group) according to the treatment agents: normal , DM (nontreated diabetic group), DM+DA-9801 (DA-9801-treated diabetic group), and DM+ALA (ALA-treated diabetic group). DA-9801 was administered orally at a dose of 100 mg/kg/day, while 0.5% ALA, which was in powdered form, was mixed with daily food at a dose of 50 mg/kg/day. On the 8th and 16th weeks of treatment, behavioral assessment and morphometric comparison were performed. On the 17th week, biochemical parameters including NGF, TNF-α, and IL-6 levels in sciatic nerve and spinal cord were measured.Six to eight-week-old male Sprague-Dawley (SD) rats weighing 160 to 180 g were purchased from Damool Science . Animals were housed in an optimal condition with a 12 hr light and dark cycle. The room temperature was maintained at 23 ± 1°C and humidity at 53 ± 2%. The animals had a free access to food and water. All experiments and protocols were performed after the approval by the Institutional Rat Care and Use Committee of the Chonbuk National University Medical School (CBU 2011-0055). Streptozotocin (STZ)  dissolved in 0.1 mol/L sodium citrate buffer (pH 4.5) was injected intraperitoneally to experimental rats (60 mg/kg body weight) to induce diabetes. Forty-eight hours after STZ injection, rats showing blood glucose levels higher than 350 mg/dL were verified to have diabetes. After subjecting rats into an overnight fasting, blood samples were drawn from the tail vein, and blood glucose levels were measured using Precision Xtra Plus . In the same manner, age-matched control rats received an equal volume of the vehicle-sodium citrate buffer (pH 4.5). DA-9801 was supplied from Dong-A Pharmaceutical , while alpha lipoic acid (ALA) was supplied from Bukwang Pharmaceutical . After 4 weeks of STZ and sodium citrate buffer injection, normal and diabetic rats were randomly selected and divided into 4 groups  to evaluate the withdrawal threshold of hind paw. After adaptation to the testing condition for at least 20 min, rats were placed individually in a plastic cage with 1 cm sized perforated mesh. Von Frey filament, with calibrated bending forces (in g), were applied perpendicularly to the plantar surface of the hind paw to deliver tactile stimuli of varying intensity. This test was conducted according to the procedure of Chaplan et al. . The stiµL of Triton lysis buffer. After centrifugation at 13,200 rpm for 15 min at 4°C, the supernatant was transferred to a fresh tube (1.5 mL capacity), and the protein concentration was quantified following Bradford method [α, and IL-6  overnight at 4°C. The membranes were washed in 1 TBST by shaking 3 times for 10 min. Incubation with HRP-conjugated secondary antibody donkey anti-goat IgG, HRP conjugate  was performed on a rocker for 1 hr at 4°C. Protein bands were detected using ECL-plus kit . Quantitative image analysis was conducted using LAS 3000 Fuji film and film densitometry was performed using MultiGauge version 3.0 (Fuji film).On the 17th week, sciatic nerves and spinal cord were rapidly removed after killing all rats under deep anesthesia and were prepared for western blot analysis. Sciatic nerve and spinal cord were broken down mechanically with 100–200 μm transverse sections were stained with toluidine blue. The procedures used for immunohistochemical analysis were the same as those described previously [μm in thickness, cut perpendicular to the dermis, were prepared with a sliding cryostat  and were immersed in PBS for 15 min at room temperature. Samples were then transferred into microtubes containing Dako Protein Block Serum Free  as a blocking buffer supplemented with 3% goat serum. After 30 min of blocking on a shaker table at room temperature, specimen sections were washed with PBS twice for 10 min and then incubated overnight with primary antibody, rabbit anti-protein-gene-product 9.5 (PGP 9.5)  at a dilution of 1 : 100 at 4°C. The antibodies were diluted in antibody diluent  supplemented with 1% goat serum. After complete washing, the specimens were incubated with the secondary antibody, goat anti-rabbit IgG-FITC , for 1 hr at room temperature in a dark room. After washing with PBS, sections were placed on slides and mounted with a fluorescent mounting media .On the 0th and 8th weeks, 3 × 3 mm tissues were taken from the dorsum of the foot via skin biopsy for immunohistochemical analyses of intraepidermal nerve fiber (IENF). On the 17th week after killing all rats under deep anesthesia, last cutaneous tissue samples from the feet and segments of the sciatic nerve were obtained from each rat. Sciatic nerve tissue samples were immersed in a fixative ) and incubated overnight at 4°C. These samples were then embedded in JB-4 solution , and 1.5 eviously . Skin tiPhotomicrographs of the myelinated fiber and IENF were captured using a digital camera  with a final magnification of 400 and 100 times, respectively. The myelinated fiber or axonal area in the sciatic nerve, represented by the outer or inner border of the myelin sheath, was measured with the aid of analySIS image software , and the mean myelinated fiber area was determined. In addition, the thickness of myelin sheath and the diameter of axonal fiber were measured.PGP 9.5-immunoreactive nerve fibers in the epidermis of each section were counted as described previously . In cutaP < 0.05 were considered as statistically significant. Statistical analyses were performed using SPSS 12.0 software .All data were expressed as means ± SD. One-way ANOVA with Duncan's post hoc test was used to compare experimental groups. The confidence interval for testing the differences was 95%, and analyses results with Two weeks after STZ injection, increased blood glucose levels and decreased body weight were observed in diabetic groups compared with normal rats. Treatment with DA-9801 for 16 weeks did not affect on the body weight and blood glucose levels in DM+DA-9801 group. There were no significant changes in body weight and blood glucose levels in the DM+ALA group Figures .P < 0.05)  . On the  < 0.05) . α and IL-6 levels increased in diabetic condition groups compared with the normal ones. These cytokines in each diabetic group were reduced after DA-9801 treatment, and the degree of reduction was similar in DM+DA-9801 and DM+ALA groups  in the non-treated diabetic group compared with those in normal ones. However DM+DA-9801 group recovered NGF level to a similar degree with the normal group. This degree of NGF recovery was more potent in DM+DA-9801 than in DM + ALA group, although ALA is not an NGF inducer . TNF-α a Figures .4(a) and The comparison of cutaneous peripheral nerve quantity showed markedly reduced IENF density in non-treated diabetic group by about 42–50% compared with normal group. Shortened and degenerated patterns of small peripheral nerve fibers were observed in diabetic groups . HoweverPeripheral neuropathy is a common disease in diabetic patients, and an estimate of more than half of all diabetic patients suffer from diverse neuropathic symptoms . DiabetiHence, in the present study, we investigated the potential of a botanical agent (DA-9801) in increasing NGF levels for the treatment of DPN. A comparative study with ALA, which is a well-known agent for the treatment of DPN, was also conducted. in vitro and in experimental animals [Various species of Dioscorea has been used as a traditional medicine for diverse diseases including metabolic disorders, inflammatory diseases, pain control, and neuropathic diseases –25. DA-9 animals , 8. Howe animals . These nIn the body weight and glucose levels, DA-9801 did not show the lowering effect in diabetic group. Of course, DA-9801 is not lowering agent for glucose or body weight, and our animal model was late state type 2 diabetic or type 1 diabetic model using STZ 60 mg/kg. Therefore, we thought that only one oral agent is difficult to lower the blood glucose in this condition, even though DA-9801 may have the glucose lowering effect. Accordingly, we thought that body weight was difficult to be affected without glucose control in this animal model because this agent could not affect on the food intake.α and IL-6, which are well known to be involved in DPN pathogenesis. In the present study, the increases of TNF-α and IL-6 in diabetic group were reduced after DA-9801 treatment. Therefore, neuronal effect mediated by increased NGF level and anti-inflammation mediated via TNF-α and IL-6 may have played a role in the peripheral nerve protection in DM+DA-9801 group. However, the degree of the contribution of NGF and cytokines on DPN treatment has not been confirmed.Anti-inflammation, antioxidant, or neuronal regeneration are fundamental mechanisms for nerve protection, although more detailed diverse pathways including several mediators are involved in DPN pathogenesis , 28. TheTo support our claim of therapeutic potential of DA-9801 for peripheral nerve protection, the structural change of cutaneous small nerve fibers and sciatic nerves was also compared among experimental groups. We found that the quantity of small intraepidermal nerve fibers was more preserved, and the pattern was less degenerated in DM+DA-9801 and DM+ALA groups than in non-treated diabetic group. Moreover, the degree of neuronal protection of DA-9801 was found to be more potent than ALA. Based on IENF density measurements, DM+DA-9801 or DM+ALA groups exhibited less degenerated morphology of myelin sheath and axon compared with the non-treated diabetic group. DA-9801 also showed more potent therapeutic potential than ALA in the present study. Taking all results as a whole, this study showed that DA-9801 can act as a biochemical and morphological protective agent on the peripheral nerves in diabetic patients. However, the potential of DA-9801 to induce other neurotrophic factors or improve other mediators involved in pathogenesis of DPN remains to be determined. Aside from NGF, brain-derived neurotrophic factor, neurotrophin- (NT-) 3, IGF-I, IGF-II, and glial cell-derived neurotrophic factors have also been examined as therapeutic agents , 32.However, this study have several limitations. First, although the NGF agonistic activity and anti-inflammatory effect of DA-9801 was proven to contribute to the prevention of peripheral nerve damage, more detailed mechanism and identification of the active components of Dioscorea species are warranted for the effective use of DA-9801 in DPN management. Second, dose-response effect of DA-9801 on diverse parameters in DPN assessment should be investigated. Third, NGF pathway, in the pathophysiologic treatment of DPN, is considered to lead nerve regeneration as well as neuronal survival. However, an examination of their differences was not performed in the present study. Further investigation on the effects of DA-9801 for the treatment of DPN should be conducted. α and IL-6 may reduce the deterioration of the peripheral nerves by chronic hyperglycemia. However, DA-9801 has not completely prevented the hyperglycemia-induced metabolic disturbance in the peripheral nerves. Therefore, multiple steps involved in the pathogenesis of DPN need further assessment to attain more effective pathogenic correction or complete prevention of DPN progression.In summary, the results of this study suggest the potential of DA-9801 for the prevention of degeneration or induction of regeneration of peripheral nerves in diabetic rats. Furthermore, this therapeutic potential is greater in DA-9801 than in ALA. The effects of DA-9801 are believed to include enhancement of neurotrophic activity and anti-inflammatory response, which are commonly involved in the pathogenesis of DPN. The improvement of NGF and decreased levels of TNF-


Although the cellular mechanisms responsible for the pathogenesis of autism are not understood, a growing number of studies have suggested that localized inflammation of the central nervous system (CNS) may contribute to the development of autism. Recent evidence shows that IL-6 has a crucial role in the development and plasticity of CNS.In vitro adenoviral gene delivery approach was used to over-express IL-6 in cultured cerebellar granule cells. Cell adhesion and migration assays, DiI labeling, TO-PRO-3 staining and immunofluorescence were used to examine cell adhesion and migration, dendritic spine morphology, cell apoptosis and synaptic protein expression respectively.Immunohistochemistry studies were employed to detect the IL-6 expression in the cerebellum of study subjects. In this study, we found that IL-6 was significantly increased in the cerebellum of autistic subjects. We investigated how IL-6 affects neural cell development and function by transfecting cultured mouse cerebellar granule cells with an IL-6 viral expression vector. We demonstrated that IL-6 over-expression in granule cells caused impairments in granule cell adhesion and migration but had little effect on the formation of dendritic spines or granule cell apoptosis. However, IL-6 over-expression stimulated the formation of granule cell excitatory synapses, without affecting inhibitory synapses.Our results provide further evidence that aberrant IL-6 may be associated with autism. In addition, our results suggest that the elevated IL-6 in the autistic brain could alter neural cell adhesion, migration and also cause an imbalance of excitatory and inhibitory circuits. Thus, increased IL-6 expression may be partially responsible for the pathogenesis of autism. Autistic disorder is the most severe of a group of neurodevelopmental disorders, referred to as autism spectrum disorders (ASDs), and is characterized by problems in communication, social skills, and repetitive behavior. Susceptibility to autism is clearly attributable to genetic factors -4, but tet al demonstrated that IL-6 promotes specific neuronal differentiation of neural progenitor cells from the adult hippocampus -buffered saline (TBS). The sections were then incubated with primary antibodies  overnight at 4°C. After washing in TBS for 5 min, the sections were further incubated with the secondary antibody  for 30 min at RT, followed by incubation in Avidin-biotinylated peroxidase (VectaStain Elite ABC Kit) for 45 min at RT and in 0.0125 g DAB/25 ml 0.05 M TBS/1 drop 30% H2O2 for 10 min at RT. All sections were dehydrated with ethanol and Xylene before mounting for examination by confocal microscopy . Image J analysis  was used to calculate areas and immunostaining densities.Cerebellar granule cells (CGCs) were prepared from C57BL/6J mouse pups  as described previously . BrieflyMouse IL-6 GFP adenovirus (IL-6 group) and GFP adenovirus (Control group) were generated by Welgen . After culturing for 4-5 days, CGCs were infected with Ad-GFP-IL-6 or Ad-GFP at multiplicity of infection (MOI) of 500. After 24 h, the medium was replaced with normal growth medium. Three days after infection, CGCs were used for the experiments reported in the present study.ueous One Solution kit, Promega).10, 000 CGCs were plated per well in 96 well tissue culture plates coated with recombinant ICAM-1  at the final concentration of 10 μg/ml. After 1.5 h of attachment, unattached cells were removed by aspiration and adherent cells were quantitated by the colorimetric aqueous MTS assay  at a final concentration of 2.5 μM. 10, 000 labeled cells were plated in 0.8 ml DMEM +1% FBS in each well of 24 well chambers adapted for the HTS Fluoroblock  apparatus. After 3 h, 5% FBS was added to the lower chamber medium to establish a 1-5% serum gradient. Migration of cells from the upper to lower chamber were quantitated at 2 h using a microfluorimetric plate reader .et al [The cultured cells were labeled using a protocol adapted from Hering et al . Brieflyet al ,28. For et al . The numTo exam the apoptotic effect of IL-6 on CGCs, TO-PRO-3 staining was used to visualize the nuclear morphology of apoptotic cell. Cells that had bright condensed, fragmented nuclei after staining were considered to be apoptotic ,31. To iCGCs were fixed in 4% formaldehyde for 15 min and blocked with 3% goat serum/0.3% Triton X-100 in PBS and incubated with anti-Syp polyclonal antibody , anti-VGLUT1 monoclonal antibody , and anti-VGAT polyclonal antibody  overnight at 4°C, followed by incubation with Alexa Fluor 577 anti-mouse and anti-rabbit IgG  for 1.5 h at room temperature. Cultures were mounted on glass slides with ProLong Gold antifade reagent (Invitrogen), and then imaged using Nikon eclipse 90i confocal laser scanning microscope.The analysis of immunofluorescence images were done as described previously . Images We determined the statistical significance among groups with the unpaired t test using the StatView 5.0 software . A null hypothesis probability of <0.05% was considered significant. All data are presented as means ± standard error (SEM).Immunohistochemistry studies were conducted to examine IL-6 expression in the cerebellum of six autistic subjects and six age matched controls using IL-6 antibody. The results are shown in Figure in vitro experiments confirmed CGCs were efficiently infected by an adenoviral vector carrying the GFP. The infection efficiency as revealed by GFP signal was over than 90%  or GFP viral vector alone  were seeded on the ICAM-1-coated surface. The efficiency of cell adhesion was evaluated by a modified MTS assay based on the dehydrogenase conversion of MTS to colored tetrazolium salt, which occurs only in viable cells that adhered to the substrate. The amount of colored product formed at 490 nm (OD 490) was proportional to the number of attached cells. As shown in Figure Cell migration is modulated by a complex of adhesion molecules between migrating cells and surrounding extracellular matrix (ECM) proteins, and critically affected by cell adhesion . Since wTo test whether over-expression of IL-6 affected dendritic spine generation, DiI labelling was employed to show their density. Control CGCs showed spines at a density of 12.40 ± 0.65 spines per 50 μm dendrite length, while IL-6 CGCs showed a density of 10.50 ± 0.80 (p = 0.08, Figure in vitro and in vivo [Granule cell apoptosis can be induced by various factors and has been demonstrated both  in vivo . A group in vivo -40. In o in vivo ,42. As sAutism has been postulated to arise from functional changes in neural circuitry and to be associated with an imbalance between excitatory and inhibitory synaptic transmission, but the mechanisms involved are unknown . To inveLocalized CNS inflammation has been implicated in the development of autism. Cytokines including TNF-α, IFN-γ, IL-1β and IL-12 have been reported to be elevated in the blood mononuclear cells, serum and plasma of autistic subjects ,20-24. RAn elevated cytokine response is associated with autism and IL-6 has been repeatedly found to be increased in the autistic brain. In this study, we developed a neural cell model of IL-6 over-expression with an adenoviral expression vector approach and examined the effects of excessive IL-6 on neural cell properties and functions. We showed that IL-6 over-expression in cultured mouse cerebellar granule and microglial cells significantly impaired neural cell migration and adhesion. Neuronal migration is a fundamental process that determines the final location of neurons in the nervous system, and thus establishes the basis for the subsequent wiring of neural circuits. From cell polarization to target identification, neuronal migration enables neuronal precursors to move across the brain to reach their final destination by integrating multiple cellular and molecular events . Thus, wEmerging evidence points to an association of apoptosis with certain neuropsychiatric disorders including autism. Araghi-Niknam and Fatemi  demonstrin vitro studies showed that increased IL-6 stimulates granule cell synapse formation, particularly enhanced excitatory synapse formation, while it had little effect on inhibitory synapses. We also demonstrated that IL-6 over-expression in granule cells affected granule cell adhesion and migration, which suggests that IL-6 could be involved in the regulation of CAMs that critically modulate excitatory synaptic formation. It will be important to further study the underlying mechanisms by which IL-6 affects the function of CAMs and the way CAMs may be regulated by IL-6. A number of studies have demonstrated that mutations in the synaptic adhesion molecules neurexin 1 and neuroligins 3 and 4 are associated with autism [Synapses are asymmetric intercellular junctions that mediate neuronal communication. The number, type, and connectivity patterns of synapses determine the formation, maintenance, and function of neural circuitries. Improper synapse formation and function may cause neurodevelopmental disorders, such as mental retardation and autism ,51, and h autism . The scah autism ,55. Thesin vitro impaired granule cell adhesion and migration. In addition, we found that IL-6 over-expression stimulated the formation of excitatory synapses of granule cells, while having no effect on the inhibitory synapses. These findings suggest that the elevated IL-6 in the autistic brain could cause an imbalance of neuronal circuits through its effects on neural cell adhesion/migration and synapse formation, and contribute to the development of autism.In conclusion, our study demonstrates that IL-6 was significantly increased in the cerebellum of autistic subjects as compared with age- and sex-matched controls. We also showed that IL-6 over-expression in cerebellar granule cells The authors declare that they have no competing interests.XL participated in the design of the study, evaluation and analyses of the results, and drafting of the manuscript. HW participated in conducting most of the experiments, statistical analyses of the results. HZ, AS and CD participated in Western blot and cell culture. MM and WTB participated in results analysis and manuscript edition. All authors read and approved the final manuscript.Table S1. Study subject information.Click here for file


Aim. To investigate whether HLA class I polymorphisms could influence the clearance of hepatitis B surface antigen (HBsAg) in Qidong Han population. Methods. We genotyped HLA-A, -B, and -C loci of 448 individuals with HBV persistent infection and 140 persons with spontaneous clearance of HBsAg by polymerase chain reaction with sequencing based typing (PCR/SBT). All the individuals were unrelated males enrolled from Qidong Han population and were followed up for 10 years. Results.The frequency of HLA-A∗33:03:01G was increased in persistent HBV infection group , while frequency of HLA-B∗13:01:01G was increased in HBsAg clearance group . Conclusion. These findings suggested that the host HLA class I polymorphism is an important factor in determining the outcomes of HBV infection. Hepatitis B virus (HBV) infection is a major public health problem. Two billion people are expected to be infected with HBV during their lifetime and about 350 million are estimated to be chronic carriers , 2. NowaHuman leukocyte antigen (HLA) is an integral component of the immune response on which majority of host genetic studies have focused. Numerous reports described the associations of highly polymorphic HLA gene with the outcome of a wide range of infectious diseases. It is because antiviral cytotoxic T lymphocytes (CTLs) are believed to play a major role in eradication of infection by virtue of their capacity to identify and kill virus-infected cells through recognition of viral peptides presented by HLA class I molecules . Thus, HThe genes of the HLA locus are located on the short arm of chromosome 6. They are arranged in three clusters: class I, class II, and class III. Many previous studies have shown the associations of certain HLA class I genes with the course of HBV infection, but these associations are inconsistent even within the same population , 11. HanThe protocol was approved by the Ethics Committee of the Qidong Liver Cancer Institute, and all patients provided written, informed consent before enrollment.A total of 728 unrelated male individuals with hepatitis B surface antigen (HBsAg) positive in sera were enrolled in the Institute of Liver Cancer of Qidong in 1995. They were followed up for 10 years and tested for HBV infection again. In 2005, 588 individuals were included in this cohort because of loss to follow-up and exclusion of the patients who were treated with anti-HBV therapy. Among these 588 persons, 448 remained HBsAg positive (named HBV persistent infection group), while 140 were negative for HBsAg but positive for both anti-hepatitis B virus core antigen (HBc) and anti-HBsAg (named HBsAg clearance group). None of the subjects were positive for hepatitis C virus (HCV), hepatitis D virus (HDV), and human immunodeficiency virus (HIV) antibody. All of them are Han population.Plasma samples were obtained from all the subjects and stored at −20°C. Their serologic status with regard to HBsAg, anti-HBs, anti-HBc, hepatitis B virus e antigen (HBeAg), and anti-HBe was determined by enzyme-linked immunosorbent assay according to the manufacturer's instructions .Genomic DNA for each individual was extracted from peripheral blood mononuclear cells using the QIAamp DNA blood kit . All DNA samples (100–200 ng/mL) were stored at 4°C (short term) or −20°C (long term) in TE buffer  and diluted to 10–12 ng/mL in PCR.http://www.ncbi.nlm.nih.gov/mhc/). All groups of HLA alleles that have identical nucleotide sequences across the exons encoding the peptide binding domains  are designated by a code, such as A*33:03:01G, which essentially contains A*33:03:01, A*33:15, A*33:25, and so on .Genomic DNA was amplified by the standard HLA locus-specific primers . AccordiP value was less than Pc (0.05/29 = 0.0017). HLA genotypes with frequency not less than 3% were checked for the Hardy-Weinberg equilibrium using χ2 test. Data were analyzed by SPSS 13.0 data analysis software package. Odds ratios (OR) and their 95% confidence intervals were calculated.The allele frequency was calculated as the number of the allele divided by the double numbers of the total samples. The significance of allelic associations was determined either by chi-square test or by Fisher's exact test (when there were less than five subjects in a cell). In order to reduce the number of comparisons with inadequate power, only alleles with frequency not less than 3%  were analyzed. The level of significance was adjusted for multiple testing using the Bonferroni correction. According to the Bonferroni correction, the frequency of HLA allele was considered to be statistically different only when uncorrected 588 Qidong Han persons who were tested as HBsAg positive in 1995 were included in this study. 448 were classified as HBV persistent infection group as they remained HBsAg positive 10 years of later. The HBsAg clearance group includes the remaining 140 individuals who were negative for HBsAg but positive for both anti-HBc and anti-HBs after 10 years follow-up. Age distribution showed no statistical difference between the two groups.B*46:01:01G and B*40:01:01G were the most frequent HLA-B alleles  were identified by our previous study from this cohort and named by the WHO Nomenclature Committee [P > 0.05).A total of 31 HLA-A alleles, 54 HLA-B alleles, and 35 HLA-C alleles were detected in the two study groups Tables , while B alleles , and C*0C allele . Meanwhiommittee , 14. HLAHLA-A*33:03:01G allele in the HBV persistent infection group was higher than that in the HBsAg clearance group , whereas the frequency of HLA-B*13 : 01 : 01G was higher in the HBsAg clearance group than that in the persistent group . However, only the frequency of HLA-B*13 : 01 : 01G increased significantly in HBsAg clearance group after the Bonferroni correction.To maintain statistical power, only those alleles with the frequencies not less than 3% were compared between the persistent infection and HBsAg clearance groups. The frequency of Cytomegalovirus (CMV) [Bw4, HLA-C group 1, and HLA-C group 2 as described [It is doubtless that host genetic factors are associated with the clinical outcome of different infectious diseases. The most-studied host factors that might affect the susceptibility to viral infection are HLA genes , 16. Amous (CMV) , supportus (CMV) . We alsous (CMV) . To inveescribed , and theHLA-A*0206 was susceptible to HBV infection and chronicity, and HLA-B*4001 was likely to be associated with the elimination of the virus [HLA-A*01-B*08-DRB1*03, B*44-Cw1601, and B*44-Cw*0501 haplotypes were associated with viral persistence, and HLA-A*0301 was associated with viral clearance. In European Caucasians, HLA-B*8 was associated with non-response to HBV vaccination [HLA-A24 and CW1 were protective against chronic HBV [HLA-B35 and HLA-CW4 were significantly higher in the chronic HBV group than those in the spontaneously recovered group [The relationship between HLA class I polymorphisms and the outcome of HBV infection has been explored previously, but this relationship does not appear to be universal since the investigated populations are different. In Taiwanese Aborigines, he virus . In Amercination . Karan eonic HBV . Besidesed group .HLA-A*33:03:01G was more frequent in the HBV persistent infection group than in the HBsAg clearance group , which was consistent with the results found in northern Iran [In this study, we investigated a cohort of Qidong Han population who lived in an area of high prevalence of HBV infection. The samples were made of unrelated males and were followed up for 10 years. No previous prospective cohort study has been conducted to clarify the relationship between the polymorphisms of HLA alleles and the outcome of HBV infection in such a population. We found that ern Iran  althoughHLA-B*13:01:01G was significantly higher in the HBsAg clearance group than that in the persistent group , which revealed that HLA-B*13:01:01G was associated with a protective effect against persistent HBV infection in this Qidong Han population. HLA-B*13:01:01G is not a KIR ligand because it does not carry a Bw4 motif. Therefore, it is unlikely that NK cell can function through this KIR-HLA interaction. Interestingly, the frequencies of HLA-B*13:02:01G were almost identical in the two groups (2.86% in HBsAg clearance group versus 3.46% in persistent infection group). HLA-B*13:02:01G differs from HLA-B*13:01:01G for only three amino acids at positions 94, 95, and 97. This three-amino-acid difference probably affects the conformation of the MHC binding grooves and results in their abilities to present viral peptide to CD8+ CTLs, which leads to different prognosis after HBV infection. This possible molecular mechanism needs to be tested in the future study.The frequency of HLA-B*13:01:01G was associated with a protective effect against persistent HBV infection in Qidong Han population. Our study provides a new clue about the influence of HLA class I diversity on the natural history of HBV infection in this population. These findings suggest that the host HLA polymorphism is an important factor in determining the outcome of HBV infection. Studies of the underlying molecular mechanisms need to be carried out in the future.In summary, we found that


Predicting the function of a protein is one of the major challenges in the post-genomic era where a large number of protein sequences of unknown function are accumulating rapidly. Lectins are the proteins that specifically recognize and bind to carbohydrate moieties present on either proteins or lipids. Cancerlectins are those lectins that play various important roles in tumor cell differentiation and metastasis. Although the two types of proteins are linked, still there is no computational method available that can distinguish cancerlectins from the large pool of non-cancerlectins. Hence, it is imperative to develop a method that can distinguish between cancer and non-cancerlectins.All the models developed in this study are based on a non-redundant dataset containing 178 cancerlectins and 226 non-cancerlectins in which no two sequences have more than 50% sequence similarity. We have applied the similarity search based technique, i.e. BLAST, and achieved a maximum accuracy of 43.25%. The amino acids compositional analysis have shown that certain residues  were preferred in cancerlectins whereas some other  were preferred in non-cancerlectins. It has been found that the PROSITE domain "Crystalline beta gamma" was abundant in cancerlectins whereas domains like "SUEL-type lectin domain" were found mainly in non-cancerlectins. An SVM-based model has been developed to differentiate between the cancer and non-cancerlectins which achieved a maximum Matthew's correlation coefficient (MCC) value of 0.32 with an accuracy of 64.84%, using amino acid compositions. We have developed a model based on dipeptide compositions which achieved an MCC value of 0.30 with an accuracy of 64.84%. Thereafter, we have developed models based on split compositions (2 and 4 parts) and achieved an MCC value of 0.31, 0.32 with accuracies of 65.10% and 66.09%, respectively. An SVM model based on Position Specific Scoring Matrix (PSSM), generated by PSI-BLAST, was developed and achieved an MCC value of 0.36 with an accuracy of 68.34%. Finally, we have integrated the PROSITE domain information with PSSM and developed an SVM model that has achieved an MCC value of 0.38 with 69.09% accuracy.http://www.imtech.res.in/raghava/cancer_pred/BLAST has been found inefficient to distinguish between cancer and non-cancerlectins. We analyzed the protein sequences of cancer and non-cancerlectins and identified interesting patterns. We have been able to identify PROSITE domains that are preferred in cancer and non-cancerlectins and thus provided interesting insights into the two types of proteins. The method developed in this study will be useful for researchers studying cancerlectins, lectins and cancer biology. The web-server based on the above study, is available at  Lectinscolipids . These glic acid . Not onllic acid .Cancerlectins are known to play various important roles in cancer metastasis -7. SeverCancerlectins are known in terms of their source, class, domain, fold class, quaternary structure and carbohydrate specificity but the method to distinguish cancerlectins from lectins or non-cancerlectin is still missing . Resultshttp://proline.physics.iisc.ernet.in/cgi-bin/cancerdb/input.cgi). After removing the proteins having 100% sequence similarity using the CD-HIT program, we obtained 385 sequences which formed the positive dataset. For a negative dataset, we searched the UniProt database (http://www.uniprot.org/) with the keyword "lectin" and a total number of 1550 non-redundant sequences were obtained. These were further filtered by excluding the sequences containing the keywords "similar", "fragment", "putative" and "probable", resulting in 891 lectin sequences. Seventy-one sequences were found to be common to cancerlectins and lectins. These sequences were then removed from lectins, reducing the number of lectins to 820. To balance the datasets, a total of 385 sequences were randomly selected from the 820 lectin sequences, to equalize the number of lectins to the initial number of cancerlectins. Furthermore, to make non-redundant datasets, the CD-HIT program was used at 50% cutoff resulting in 178 cancerlectin and 226 non-cancerlectin sequences.We downloaded 509 cancerlectin protein sequences from cancerlectinDB database  and reduced the redundancy up to 50% by using CD-HIT program, there were chances of similarity between the two datasets (subsets). To determine the similarity between cancer and non-cancerlectins, we employed the BLAST tool using the non-cancerlectins as test sequences against a database of cancerlectins with an E value cut-off of 0.001. Out of 226 queries, a total of 145 hits were found, which confirmed that there was 64.15% sequence similarity between non-cancerlectin and cancerlectin datasets.Evaluation of newly-developed methods is a big challenge in Bioinformatics. One of the most common techniques for model evaluation is the Jack-knife test or leave-one out cross-validation (LOOCV) -25. In tA set of parameters used to evaluate the performance of the various methods is briefly described below.Sensitivity, or percentage coverage, is the percentage of cancerlectins correctly predicted as "cancerlectins".Percentage of non-cancerlectins correctly predicted as "non-cancerlectins".Percentage of overall correctly predicted proteins (cancer and non-cancerlectins).It is the statistical parameter used to assess the quality of predictions and to correct the imbalance in the data. It is calculated as follows:versus the false positive rate (FP/FP+TN). The area under the curve (AUC) gives a single value to evaluate the performance of a method. We used the SigmaPlot 11.0 package for plotting the ROC and calculating the AUC.Where TP is the number of correctly predicted proteins in the positive dataset (cancerlectins) and TN is the number of correctly predicted proteins in the negative dataset (non-cancerlectins), whereas FP is the number of wrongly predicted proteins in the positive dataset and FN is the number of wrongly predicted proteins in the negative dataset. For the evaluation of a new prediction method MCC is considered the most robust parameter . An MCC http://www.cs.cornell.edu/People/tj/svm_light/. SVM is based on the structural risk minimization principle of statistics learning theory [light version 6.01 package of SVM and learning was carried out by using three  kernels [In this study we employed a highly successful machine learning technique known as "Support Vector Machine", which is freely available at g theory . SVM is  kernels . SVM tak kernels . In thisThe aim of calculating the composition of proteins is to convert the variable length of protein sequences into fixed length feature vectors. This is a crucial step because the SVM machine learning technique requires fixed length patterns.Amino acid composition is the fraction of each of amino acid in a protein sequence and provides vector of 20 dimensions. The SVM was provided with these 20 dimension vectors encapsulating the amino acid composition of proteins.× 20) features, one for each dipeptide. The dipeptide composition incorporates the fraction of amino acids as well as their local order i.e order of amino acids in a protein sequence. In this way, dipeptide composition is more informative than amino acid composition.Dipeptide composition was used to give global information about each protein sequence and it gives a fixed length pattern of 400  two parts (N = 2) and (ii) four parts (N = 4) generating 40 and 80 input dimensions, respectively.Split composition was used to detect conserved residues or signal peptides in any terminal of the given protein sequences -34. In c× M, where M is the length of the target sequence and each element represents the frequency of occurrence of each of the 20 amino acids [In this study, PSSM profiles were generated using PSI-BLAST  to searcno acids .× M) was scaled to the range of 0-1 using a sigmoid function. Further, in order to obtain an input of fixed length, these normalized PSSMs (20 × M) were used to generate a 400-dimensional input vector by summing all rows in the PSSM corresponding to each type of amino acid in the sequence. Finally, each element in the input vector was divided by the length of the protein sequence resulting in a matrix of 400 (20 × 20) elements, which was used as input vector for training the SVM.Next, each element of the matrix  is a Perl based stand-alone tool that combines different protein signature-recognition methods into a single platform . PROSITEhttp://www.imtech.res.in/raghava/copid/) web server. As shown in Figure We analyzed the amino acid composition of both cancer and non-cancerlectins proteins with the help of the Copid  approach by dividing the protein into two and four equal parts and calculating the amino acid compositions. This approach achieved MCC values of 0.31 and 0.32 (with accuracies 65.10% and 66.09%), respectively [Table  and 4 apIt has been shown in several studies the evolutionary information obtained using multiple sequence alignment provides more comprehensive information about a protein than a single sequence . EarlierWe selected the 14 most distinguishable domains present in either cancer or non-cancerlectins datasets: four  in cancerlectins and ten  in non-cancerlectins [Table We generated a vector of 414 dimensions which contains 400 PSSM features and 14 features for the selected 14 PROSITE domains. Finally, a SVM-based classifier was developed using 414 features, 400 from PSSM profile and 14 from domains, which achieved an accuracy level of 69.09% with MCC value of 0.38 Table  , which are growing at a tremendous rate . In the past, predictions of (I) Sugar-binding sites on proteins (II) Secondary structure of various legume lectins have been reported -44. The In this study, a systematic attempt has been made to predict cancerlectins. In amino acid composition, we collected information about the frequency of the 20 types of amino acids and used it in machine learning technique. However, this approach provides information only about the amino acid frequency, but not about the local order of amino acids. To provide information about both frequency and local order of amino acids, dipeptide composition can be used as input. To check the presence of any signal peptide present in cancerlectins, we used the split amino acid composition in the form of SVM input vectors. In our composition-based SVM models, the overall accuracy of amino acid, dipeptide, split-based compositions were comparatively similar (~65%) with MCC values of 0.32, 0.30, 0.31 respectively. The PSSM-based evolutionary information provides better information  hence weThis work attempts to predict cancerlectins, from a pool of non-cancerlectins or simple lectins. We analyzed the protein sequences of cancer and non-cancer lectins and selected the distinguishable patterns e.g. amino acid, dipeptide and split compositions. The patterns are based on evolutionary information obtained by PSSM, and PROSITE domain with PSSM. We used these patterns as input features in SVM, a machine learning technique used for classification and regression studies. We were able to model an efficient classifier from PROSITE-PSSM based approach. A web server CancerPred has been developed on the SVM modules.AI: Artificial Intelligence; AUC: Area Under Curve; BLAST: Basic Local Alignment Search Tool; MCC: Matthew's Correlation Coefficient; PSI-BLAST: Position Specific Interactive BLAST; PSSM: Position Specific Scoring Matrix; SVM: Support Vector Machine.The authors declare that they have no competing interests.RK created the dataset and developed the SVM models used for developing the web server. GPSR conceived the project, coordinated it and refined the final manuscript drafted by RK, BP and JSC. Web server was developed by RK and BP. All authors have read and approved the manuscript.Supplementary Tables. Table S1: p-values for compositional differences in cancerlectins and non-cancerlectins residues. The amino acid compositions of cancer and non-cancerlectins and p-value of composition difference in between the two types of proteins. The bold values show the significant difference in composition of cancer and non-cancerlectins, in term of p-values. Table S2: Performance of BLAST on individual test sets of cancerlectins at E-value cutoff of 0.001. The result of BLAST search on dataset of cancerlectins. The total hits means the total number of hits found for a test set in BLAST search, no hits is the number of proteins that did not get any hit whereas correct hits shows the proteins whose top most hit belongs to the cancerlectin class. The percentage coverage indicates the proteins that were predicted as cancerlectins from the BLAST search. Table S3: The performance of SVM model (Learning Parameter: -z c -t 2 -g 0.01 -c 0.5 -j 1) using Amino acid composition method. This table describes the performance of amino acid composition based SVM model at each threshold (-1 to 1), providing sensitivity, specificity, accuracy and standard error and MCC. Table S4: The performance of SVM model (Learning Parameter: -z c -t 2 -g 0.001 -c 5 -j 1) using Dipeptide composition method. This table describes the performance of dipeptide composition based SVM model at each threshold (-1 to 1), providing sensitivity, specificity, accuracy and standard error and MCC. Table S5: The performance of SVM model (Learning Parameter: -z c -t 2 -g 0.001 -c 1 -j 1) using Split amino acid composition (2-part) method. This table describes the performance of split amino acid (2-part) composition based SVM model at each threshold (-1 to 1), providing sensitivity, specificity, accuracy and standard error and MCC. Table S6: The performance of SVM model (Learning Parameter: -z c -t 2 -g 0.0001 -c 1 -j 1) using Split amino acid composition (4-part) method. This table describes the performance of split amino acid (4-part) composition based SVM model at each threshold (-1 to 1), providing sensitivity, specificity, accuracy and standard error and MCC. Table S7: The performance of SVM model (Learning Parameter: -z c -t 2 -g 7 -c 1 -j 1) using PSSM-based method. This table describes the performance of PSSM based SVM model at each threshold (-1 to 1), providing sensitivity, specificity, accuracy and standard error and MCC. Table S8: The performance of SVM model (Learning Parameter: -z c -t 2 -g 7 -c 5 -j 1) using PSSM-PROSITE Domain based method. This table describes the performance of PSSM-PROSITE domain based SVM model at each threshold (-1 to 1), providing sensitivity, specificity, accuracy and standard error and MCC. Table S9: All reported PROSITE domains in cancer and non-cancerlectins. All reported domains reported in cancer and non-cancerlectins with their frequency of occurrence. A total of 151 and 200 PROSITE domains were reported in cancer and non-cancer lectins respectively. Table S10: The performance of amino acid composition based SVM model (Learning Parameter: -z c -t 2 -g 0.01 -c 0.5 -j 1) using random dataset of cancer and non-cancerlectins. This table describes the performance of amino acid composition based SVM model at each threshold (-1 to 1), providing sensitivity, specificity, accuracy and MCC.Click here for file


Elevated blood pressure (BP) is a major risk factor for the progression of chronic kidney disease (CKD). However, little is known about the influence of prehypertension on CKD. In this study, we investigated the relationship between prehypertension and CKD in a middle-aged Korean population. Furthermore, we prospectively evaluated the effect of active BP control on deterioration of kidney function during the two-year follow-up.2. A multivariable logistic regression model was used to identify associations between BP and CKD.The Korean Genome and Epidemiology Study is a community-based prospective cohort study started in 2001, with a follow-up survey conducted every two years. A total of 9509 participants aged 40–69 years were included in a baseline study. BP was classified according to the Seventh Report of the Joint National Committee on High BP (JNC-7) categories and CKD was defined as the presence of proteinuria or eGFR< 60mL/min/1.73mThe overall prevalence of CKD was 13.2%, and significantly increased with BP level. The multivariable-adjusted odds ratio of CKD was 1.59 for prehypertension and 2.27 for hypertension, compared with a normal BP. At the two-year follow-up, among the participants with prehypertension, subjects whose BP was poorly controlled had a significantly higher risk of eGFR drop , as compared to controls. The prevalence of eGFR drop was 57.8% in the controlled BP group and 66.0% in the poorly-controlled BP group.Prehypertension, as well as hypertension, is significantly associated with CKD among middle-aged Koreans. Our results indicate that active control of the blood-pressure of prehypertensive individuals is needed to prevent deterioration of kidney function. Chronic kidney disease (CKD) is associated with all-cause mortality, progression to end-stage renal disease (ESRD), cardiovascular disease (CVD), and many chronic diseases, such as diabetes-5.2 for at least three months; in addition, GFR is usually estimated from serum creatinine (Scr) values alone or prediction equations that take into account the Scr, age, gender and race[The prevalence of CKD in the United States has increased from 10.0% in 1988–1994 to 13.1% in 1999–2004. In a poet al. found that a BP above 110/75 mmHg was significantly associated with albuminuria in a Japanese population without hypertension, but not with eGFR[Elevated blood pressure (BP) is closely associated with CKD progression, and lowering of BP may slow down GFR decline,11. A nuith eGFR.Although several studies have suggested that hypertension is a significant risk factor for CKD, little is known of its association with prehypertension and CKD. In this study, we investigated the relationship between prehypertension and CKD in a middle-aged Korean population. To determine the effect of BP control on deterioration of kidney function, we prospectively evaluated eGFR drop according to the change of BP during the two-year follow-up.2), 9509 participants were included for baseline study. At the 2-year follow-up examination, 8603 participants were enrolled in this study after exclusion of 92 death and 814 lost to follow-up. Of those, 8149 people with complete data were selected to participate in a follow-up survey. We excluded 2108 and two participants with missing Scr and BP values, respectively. Thus, 6039 subjects were included in the present study . We obtained baseline and 2-year follow-up data from the Center for Genome Science in the National Institute of Health, Korea. The details of the present cohort have been described elsewhere,19. Of 1Measurement of BP was performed after a 5 min period of rest using a mercury sphygmomanometer, and two BP readings were taken from both arms at 30 s intervals. The average systolic and diastolic BP values of two measurements taken in a sitting position were recorded.BP was classified according to the Seventh Report of the Joint National Committee on High BP (JNC-7) categories; normal Controlled BP was defined as participants who maintained a normal BP, and poorly-controlled BP as those with prehypertension or hypertension at the two-year follow-up.Demographic, medical and behavioral characteristics were obtained using a standardized questionnaire, and included age, sex, cigarette smoking and alcohol intake.Body weight and height were measured to the nearest 0.1 kg or 0.1 cm and body mass index (BMI) was calculated as body weight (kg) divided by height (meters) squared. Waist circumference (WC) was measured at the midpoint between the ribs and the iliac crest in the standing position.Blood samples were collected after an 8–14 h fast. Fasting plasma glucose (FPG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and Scr levels were measured enzymatically, and low-density lipoprotein cholesterol (LDL-C) levels were estimated by the Friedwald formula. Protein2 for at least three months[2. CKD stages were classified as follows: stage 1, proteinuria (≥ 1 +) with eGFR ≥ 90 mL/min/1.73 m2; stage 2, proteinuria (≥1 +) with eGFR 60–89 mL/min/1.73 m2; stage 3, eGFR 30–59 mL/min/1.73 m2, and stage 4, eGFR 15–29 mL/min/1.73m2. eGFR was calculated using the following equation[-1.154 × Age- 0.203 × . Changes in kidney function were evaluated using baseline and two-year follow-up eGFR values. eGFR drop denotes a decline in eGFR between baseline and the two-year follow-up.According to the Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines, CKD is defined as a marker of kidney damage and/or GFR < 60 mL/min/1.73 me months. We defi1C (HbA1C) ≥6.5%, or use of oral hypoglycemic agents using the 2010 criteria of the American Diabetes Association (ADA)[DM was diagnosed as an FPG ≥126 mg/dl or 2-h post-challenge plasma glucose (2 h PG) ≥200 mg/dl or hemoglobin Aon (ADA).P <0.05 were considered to be statistically significant. The data were analyzed using the SAS software .The baseline characteristics of subjects were expressed as means and standard deviations for continuous variables. Statistical differences between groups were performed by one-way analysis of variance (ANOVA) for continuous variables and the chi-square test for categorical variables. A multivariable logistic regression model was used to identify associations between BP and CKD. C-statistics were calculated to compare the ability of different logistic models to discriminate participants with and without CKD. After two-year follow-up, the odds ratio of eGFR drop according to BP change was calculated by multivariable logistic regression analysis. Values of 2. The BMI, WC, FPG, TC, LDL-C and TG levels were increased when BP level was high. Hypertensive participants were more likely to have a history of DM (11.5% in the prehypertension group and 19.3% in the hypertension group). Of all subjects, 25.8% were current smokers and 47.7% were alcohol drinkers. The mean GFR estimated using the Modification of Diet in Renal Disease (MDRD) equation was 74.0 ± 14.0 mL/min/1.73 m2, and was lowest in the hypertension group. The proportion of individuals with eGFR <60 mL/min/1.73 m2 in the normal BP, prehypertension and hypertension groups were 5.6%, 11.4% and 23.3%, respectively. Two-hundred and seventeen subjects (2.3%) had proteinuria (≥1+), which was more prevalent in subjects with hypertension than in those with a normal BP.The baseline characteristics of the participants according to BP category are shown in TableP trend <0.001). CKD prevalence according to stage was 13.8% in stages 1 and 2, 85.4% in stage 3, and 0.8% in stage 4. Participants with hypertension showed the highest CKD prevalence. There was a gender difference in CKD prevalence . Females had a higher prevalence of CKD in all categories  equation, the meaWe found that CKD prevalence increased with BP level; normotensive subjects showed the lowest prevalence and hypertensive subjects the highest .Hypertension is a well-known risk factor for CKD progression-14. HoweSeveral cohort studies provided strong epidemiologic evidence regarding the risk of ESRD across the BP categories. Our results were consistent with those of an earlier study, which reported that the risk of ESRD was increased even with normal but not optimal and high-normal levels, as well as hypertension,33. In oOur follow-up results showed that subjects with poorly-controlled BP were more likely to have a decreased eGFR compared with those with controlled BP. The risk of eGFR drop was significantly increased among the poorly-controlled BP group in all, and prehypertensive participants Table. These fInterestingly, the risk of CKD significantly increased in prehypertensive subjects, and those with poorly controlled BP had a higher risk of eGFR drop at the two-year follow-up. That is, the increased CKD risk begins at a near-normal BP; therefore, those individuals may be considered for active BP control to reduce the burden of kidney disease.The strength of this study was that it was a prospective community-based cohort study which evaluated the association between BP and CKD across the range of BP.2 or kidney damage for at least three months; therefore, Scr and proteinuria should be evaluated more than once. Thus, there might be an underestimation or overestimation of the prevalence of CKD in both males and females. Third, the MDRD equation has not been validated in the Korean population. Therefore, eGFR calculation using the MDRD equation might be inaccurate. Fourth, each participant’s BP measurement was based on only two readings of a single day. Also, we only evaluated in a state of BP control with BP values and self-reported medication at a baseline and 2-year follow-up. Therefore, the misclassification of BP may be possible and it might be underestimated or overestimated the prevalence of controlled BP and poorly-controlled BP.However, there were also some limitations to this study. First, the study population consisted only of those aged 40–69 years. Nevertheless, our results may be representative of the general population in Korea because the data were from a community-based prospective cohort study, which may have minimized the sampling bias effect. Second, Scr and proteinuria were measured only once. According to the K/DOQI guidelines, CKD is defined as a GFR <60 mL/min/1.73 mPrehypertension, as well as hypertension, is significantly associated with CKD among middle-aged Koreans. Our results indicate that active control of the blood-pressure of prehypertensive individuals is necessary to prevent deterioration in kidney function. Therefore, we suggest that active blood-pressure control may play an important role in prevention of CKD.1C: Hemoglobin A1C; ADA: American Diabetes Association ; ANOVA: One-way Analysis Of Variance ; MDRD: Modification of Diet in Renal Disease ; SBP: Systolic Blood Pressure ; DBP: Diastolic Blood Pressure .BP: Blood Pressure ; CKD: Chronic Kidney Disease ; ESRD: End-Stage Renal Disease ; CVD: CardioVascular Disease ; KDOQI: Kidney Disease Outcomes Quality Initiative ; eGFR: Estimated Glomerular Filtration Rate ; Scr: Serum creatinine ; KEEP: Kidney Early Evaluation Program ; NHANES: National Health and Nutrition Examination Survey ; BMI: Body Mass Index ; WC: Waist Circumference ; FPG: Fasting Plasma Glucose ; TC: Total Cholesterol ; TG: Triglyceride ; HDL-C: High-Density Lipoprotein Cholesterol ; LDL-C: Low-Density Lipoprotein Cholesterol ; DM: Diabetes Mellitus ; 2 h PG: 2-h Post-challenge plasma Glucose ; HbAThe authors declare that they have no competing interests.MJK and HYP participated in the design of the study, performed the statistical analysis and participated in drafting the manuscript. NKL performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/960/prepubMean GFR according to different estimating equations.Click here for filePrevalence of CKD according to the CKD-EPI equation.Click here for filePrevalence of CKD according to the MDRD equation with the Korean coefficient.Click here for filePrevalence of CKD according to the novel equation for Korean.Click here for file


C. leptum group, B. longum and B. breve, while formula-feeding promoted that of Bacteroides fragilis group, C. coccoides-E. rectale group, E. coli and B. lactis. Moreover, increased numbers of B. fragilis group and Staphylococcus spp., and reduced numbers of Bifidobacterium spp. and B. longum were detected in infants with increased genetic risk of developing CD. Analyses within subgroups of either breast-fed or formula-fed infants indicated that in both cases increased risk of CD was associated with lower numbers of B. longum and/or Bifidobacterium spp. In addition, in breast-fed infants the increased genetic risk of developing CD was associated with increased C. leptum group numbers, while in formula-fed infants it was associated with increased Staphylococcus and B. fragilis group numbers. Overall, milk-feeding type in conjunction with HLA-DQ genotype play a role in establishing infants' gut microbiota; moreover, breast-feeding reduced the genotype-related differences in microbiota composition, which could partly explain the protective role attributed to breast milk in this disorder.Interactions between environmental factors and predisposing genes could be involved in the development of coeliac disease (CD). This study has assessed whether milk-feeding type and HLA-genotype influence the intestinal microbiota composition of infants with a family history of CD. The study included 164 healthy newborns, with at least one first-degree relative with CD, classified according to their HLA-DQ genotype by PCR-SSP DQB1 and DQA1 typing. Faecal microbiota was analysed by quantitative PCR at 7 days, and at 1 and 4 months of age. Significant interactions between milk-feeding type and HLA-DQ genotype on bacterial numbers were not detected by applying a linear mixed-model analysis for repeated measures. In the whole population, breast-feeding promoted colonization of  Celiac disease (CD) is a chronic inflammatory disorder of the small intestine that presents in genetically predisposed individuals following gluten consumption cis or trans forms. The susceptibility to CD is increased in homozygous subjects with a cis haplotype or possessing a second HLA-DQB1*02 allele The major genetic risk factor in CD is represented by Human Leukocyte Antigen (HLA)-DQ genes. Several studies have documented that the HLA-DQA1*05 and DQB1*02 alleles, encoding for particular DQ2 molecules, confer high susceptibility to CD Bifidobacterium spp. predominate, representing up to 90% of the total faecal microbiota, whereas in formula-fed infants the microbiota is more heterogeneous Lactobacillus and BifidobacteriumBifidobacterium spp. Gut colonization starts immediately after birth and depends on multiple factors such as the type of delivery, contamination from the environment, the type of milk-feeding and, possibly, the genotype The objective of this study was to assess the gut microbial colonization process during the first 4 months of life in breast-fed and formula-fed babies at risk of developing CD, by using quantitative PCR (qPCR). The ultimate purpose of our research is to gain a better understanding of the effects of early events leading to the acquisition of intestinal microbiota and their interactions with predisposing genes on CD risk.A prospective observational study was carried out with a cohort of 164 healthy full-term newborns recruited between June 2006 and November 2010, who had a first-degree relative affected by CD. Data of mode of delivery, size, weight, weeks of gestation and type of feeding were recorded at birth and over the study period . InfantsAllSet+™ SSP or Olerup SSP™) containing nucleotides (200 µmol L−1 each), PCR buffer, 5% glycerol and 100 µg mL−1 cresol red, 0·25 µmol L−1 of each allele- or group-specific primer pair and 0·1 µmol L−1 of internal positive control primer pair matching a segment of the human growth hormone gene in a final volume of 10 µL. An initial denaturation step at 94°C for 2 min was followed by 10 two-temperature cycles (94°C for 10 s and 65°C for 60 s) and 20 three-temperature cycles . Detection of amplified alleles was carried out on 2% agarose gels after ethidium bromide staining. HLA-DQA1 alleles were genotyped in a stepwise fashion for high resolution typing to hone the risk classification of each individual.DNA was extracted from yugal mucosa cells by scraping the inner side of the infants' cheek with sterile swabs  and purified according to the DNA IQ™ Casework Sample Kit for Maxwell® 16 protocol . Low-resolution HLA-DQB1 typing was performed by PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primers) analysis Stool samples were collected from every subject at 7 days, 1 month and 4 months of age and frozen at −20°C immediately. Samples (1 g) were diluted 1∶10 (w/v) in PBS (pH 7.2) and homogenized by thorough agitation in a vortex. Aliquots were used for DNA extraction using the QIAamp DNA stool Mini kit  following the manufacturer's instructions. DNA extractions from different pure cultures of reference strains were done following the same protocol.2 to 109 cells, as determined by microscopy counts after staining with 4′, 6 diamino-2-phenylindole in an epifluorescence microscope .qPCR was used to quantify the different bacterial groups of the faecal microbiota using genus-, group- and species-specific primers as previously described post hoc LSD test for continuous variables. Microbiological data were transformed from exponential numbers into logarithms to adjust to normal and, in the tables, are expressed as mean values of log cells/g faeces and standard deviations (SDs). A mixed model with repeated measures with three fixed factors  was applied to determine the effects of genetic risk and type of feeding on bacterial counts. Interactions  between these three factors were also analysed and not detected. Interactions between faecal microbial counts and type of delivery were not found and, therefore, data from infants with different type of delivery were grouped for statistical analyses. Differences between bacterial numbers at each sampling time (age) were analysed by ANOVA and post hoc LSD test. Correlations between factors  were determined by Pearson correlation coefficients and correlations between bacterial counts and type of milk-feeding were tested by applying the Chi-Square Pearson test. In all cases, p-values less than 0.050 were considered statistically significant.Data were analysed using the SPSS 19.0 software for Windows . The demographic characteristics of the study subjects are given as mean values (standard deviations [SDs]) for continuous variables and as numbers and proportions for categorical variables. Differences in demographic characteristic measures between the study groups were assessed using the Chi-Square Pearson test for categorical variables and ANOVA and The demographic characteristics of the infants under study are shown in cis (DQA1*0501-DQB1*0201 in homozygosis) and trans conformations (DQA1*0201-DQB1*0202 with DQA1*0505-DQB1*0301 in heterozygosis). The second group included those subjects carrying the DQ2 haplotype in cis conformation along with any other haplotype, as well as subjects carrying the DQ8 haplotype (DQA1*0301-DQB1*0302) in homozygosis. The third group included those individuals with other common genotypes not associated with CD. Of the 164 infants under study, 28.40% were classified in the first risk group, with the highest probability (>20%) of developing CD (the high genetic risk group) and 42.59% in the second group, with a >7% probability of developing CD (the intermediate genetic risk group). The remaining 29.01% of infants comprised the third group, with the lowest probability (<1%) of developing CD (the low genetic risk group).Infants were classified into three main groups according to their HLA-DQ genotype, and probabilities of developing CD were estimated according to previous studies C. leptum group (P = 0.005), B. longum (P = 0.050), and B. breve (P = 0.008) numbers were significantly higher in breast-fed than in formula-fed infants, whereas Bacteroides fragilis group (P = 0.004), C. coccoides-E. rectale group (P<0.001), E. coli (P = 0.026), and B. lactis (P = 0.002) numbers were higher in formula-fed infants than in breast-fed infants.The effect of milk-feeding type on the composition of the faecal microbiota, irrespectively of genotype, was evaluated over the study period. The type of milk feeding significantly influence different bacterial group counts by analysing data with the linear mixed model with time sampling as the repeated measure. C. leptum group and B. breve correlated with breast-feeding at 7 days of age . Moreover, formula-feeding correlated with increased numbers of E. coli, C. coccoides-E. rectale group and B. lactis at 1 month of age , and with increased numbers of C. coccoides-E. rectale and Bacteroides fragilis groups at 4 months of age .Correlations between milk-feeding type and several bacterial group counts at each sampling time were also analysed. Increased numbers of Bacteroides fragilis and C. coccoides-E. rectale group counts and longer breast-feeding duration .When analysing the cumulative effect of breast-feeding on the microbiota composition of 4-month-old infants, statistically significant negative correlations were established between increased Bifidobacterium spp. (P<0.001) and B. longum (P<0.001), whose numbers increased when genetic risk of CD decreased. The effect of genetic risk was also significant for Staphylococcus spp. (P = 0.010) and Bacteroides fragilis group (P = 0.050), whose counts were higher when infants' genetic risk was greater. The influence of HLA-DQ genotype on fecal microbiota composition at each sampling time  is also shown in The influence of HLA-DQ genotype, irrespective of milk-feeding type, on fecal microbiota composition was established by applying a linear mixed-model analysis with sampling time as the repeated measure. According to this analysis, the effect of the genetic risk on bacterial numbers was significant for Bifidobacterium spp.  and B. longum  and reduced genetic risk during the whole sampling period. In contrast, increased numbers of Staphylococcus spp. at 1 and 4 months  and B. lactis at 4 months  correlated with increased genetic risk.Correlations between genetic risk and several bacterial group counts at different infant ages (sampling time) were also established. Statistically significant correlations were found between increased numbers of both Bifidobacterium spp. (P = 0.046), which decreased when the genetic risk was increased, and on bacterial counts of Staphylococcus spp. (P = 0.030), C. leptum group (P = 0.047), B. adolescentis ((P = 0.028) and B. dentium (P = 0.009), which increased when the genetic risk was also increased, according to linear mixed-model analysis with sampling time (age) as the repeated measure. The differences in mean bacterial numbers according to the genetic risk of developing CD in breast-fed infants analysed at each sampling point are also shown in The microbiota composition of infants grouped according to milk-feeding type was also analysed as a function of HLA-DQ genotype over the study period to eliminate the effects of milk-feeding type. In breast-fed infants, the effect of genetic risk of CD was significant on bacterial counts of  P = 0.04, which d P = 0.04, B. adolC. leptum group counts in breast-fed infants .Correlations between genetic risk and bacterial counts were also analysed and these were significant between increased genetic risk of CD development and increased Bifidobacterium spp. and B. longum counts was found significant  by applying a linear mixed model analysis with sampling time as the repeated measure, and those infants with reduced genetic risk had increased numbers of these bacterial groups. The effect of genetic risk on numbers of B. fragilis group and Staphylococcus spp. was also significant, following the opposite trend . The differences in mean bacterial numbers according to the genetic risk of developing CD in formula-fed infants analysed at each sampling point are also shown in In formula-fed infants, the effect of genetic risk on Bifidobacterium spp. correlated with increased genetic risk of developing CD . At 1 month, reduced numbers of Bifidobacterium spp. and B. longum correlated with increased genetic risk of developing CD , and increased numbers of Staphylococcus spp. correlated with increased genetic risk of developing CD . At 4 months, reduced numbers of Bifidobacterium spp. and B. longum correlated with increased genetic risk of developing CD , and increased numbers of Staphylococcus spp. and B. lactis correlated with increased genetic risk .Statistically significant correlations were also established between the genetic risk of CD development and bacterial group numbers in formula-fed infants at different sampling times. At 7 days, reduced numbers of Bifidobacterium spp., and B. longum were detected . Similarly, when infants were breast-fed for more than 1 month and less than 4 months, low numbers of Bifidobacterium spp. correlated with high genetic CD risk , whereas high B. lactis numbers correlated with high genetic CD risk . On the other hand, when breast-feeding was exclusive until the fourth month of age, the high genetic risk correlated with increased numbers of C. leptum group .The cumulative effect of breast-feeding on microbiota composition was also evaluated at 4 months of age and some significant correlations were established. When infants had never been breast-fed, significant correlations between increased genetic risk and low numbers of C. leptum group, B. longum and B. breve gut colonization, while formula-feeding favoured that of B. lactis, E. coli, C. coccoides-E. rectale group and B. fragilis group. Similar trends were detected when considering the cumulative effect of breast-feeding particularly for C. coccoides-E. rectale and B. fragilis groups.This is the first report on the effects of both milk feeding type and HLA-DQ genotype on the gut microbial colonization process of healthy full-term infants with a family history of CD. In the present study, milk-feeding type influenced the composition of the microbiota, partially in agreement with previous studies Bifidobacterium spp. and B. longum were characteristic of microbiota of infants with the lowest genetic risk, whereas increased numbers of Staphylococcus spp. and B. fragilis group were characteristic of that of infants with the highest genetic CD risk. To date, there is limited evidence of a correlation between genotype and intestinal microbiota composition in humans. In previous human studies, monozygotic twins were demonstrated to have more similar faecal bacterial DNA profiles than unrelated individuals Staphylococcus counts detected in the high CD genetic risk group of infants could favour the activation of a robust T-cell response by the preferential interaction of certain superantigens, such as staphylococcal superantigen A, with HLA-DQ molecules, thereby enhancing the risk of T-cell mediated diseases In addition, specific features of fecal microbiota were associated with the genetic risk of developing CD, based on HLA-DQ genotype, when considered irrespectively of milk-feeding type. Increased numbers of B. fragilis group, which were also higher in infants with higher genetic risk of developing CD in the whole population and in the subgroup of formula-fed infants, suggesting that the colonization of this bacterial group is greatly defined by type of milk-feeding. Nevertheless, increased counts of Staphylococcus spp. were associated with increased genetic risk of developing CD in the whole population and in both breast- and formula-fed infant subgroups, but their colonization was not favoured by formula feeding, suggesting that the HLA-DQ genotype plays a more prominent role in the colonization of this bacterial group. Notably, reduced numbers of Bifidobacterium spp. were associated with an increased risk of developing CD in the whole population and in both breast- and formula-fed infants, and the colonization of species of this genus was also favoured by breast-feeding. Therefore, the findings suggest that bifidobacterial numbers can be influenced by both the HLA-DQ genotype and the milk-feeding type. Moreover, low counts of the species B. longum were found in infants of higher risk to develop CD in the whole population and in the subgroup of formula-fed infants but not in the subgroup of breast-fed infants indicating that the breast-feeding is providing to the infant's gut microbiota certain Bifidobacterium spp. The influence of the HLA-DQ genotype was also analysed in subgroups of either breast-fed or formula fed infants to gather more information about the respective effects of each variable (genotype and milk-feeding type) on the microbiota of the infants under study. In the whole infant population, formula feeding favoured the presence of increased numbers of stimuli for the adequate development of immune functions and oral tolerance Bifidobacterium to Clostridium counts in the faecal microbiota of infants was shown to precede the development of atopic diseases later in life, indicating that the relative proportions of these bacterial groups may favour or protect against the development of immune-related disorders Bifidobacterium spp. and B. longum levels in both biopsies and faeces have been reported lower in CD patients than in healthy controls C. leptum group when this factor was considered alone, and this bacterial group was associated with an increased risk of developing CD in the subgroup of breast-fed infants. C. leptum group numbers were also reported to be higher in the fecal and duodenal microbiota of CD patients than in healthy controls These and other commensal bacteria are recognized as constituting major Overall, this study demonstrates that the milk-feeding type and the HLA-DQ genotype differently influence the bacterial colonization pattern of the newborn intestine during the first 4 months of life and, therefore, could also influence the risk of developing CD in later life. Breast-feeding reduced the genotype-related differences in microbiota composition, which could partly explain the protective role attributed to breast milk in this disorder. Further studies are underway to reveal additional evidence of the role played by early intestinal colonization patterns in CD development in this cohort of infants.